The development of a diagnostic test for the detection of a specific type of microorganism in a test sample is a very slow and difficult process due to several factors. First, to develop a diagnostic test for a specific microorganism one must identify a way to detect it in a manner which is highly sensitive and easily recognizable. Generally, this is accomplished by incorporating a detection molecule into the growth medium that interacts with the target microorganism biochemically to produce a measurable signal. The best type of signals are those which result in a color change of the growth medium. Second, the signal should not interact with the matrix of the test sample resulting in a false positive reaction. This is particularly problematic if the test sample is derived from a living or once living entity such as a food product because such entities possess many of the same biochemical processes of a target microorganism. Finally, most test agents, for example a raw food product, can be contaminated by literally billions of microorganisms of differing types per gram of food. As a result, a useful diagnostic test should possess a high selectivity for the target microorganism. This is generally accomplished by either incorporating a “cocktail” of antimicrobial agents into the growth medium to suppress the growth and detection of non-target microorganisms or by selecting a detection molecule that is not recognized by a vast majority of non-target microorganisms.
Microbial growth indicators normally react chemically with a metabolic by-product produced by target microbes resulting in a color change in the medium. Examples of chemicals which changes color in the presence of pH changes associated with microbial growth include aniline blue, phenol red, bromocresol blue, and neutral red. For example, Gibson, U.S. Pat. No. 4,140,580 uses aniline blue, a chemical which changes color in the presence of acidic metabolic waste products produced by yeasts.
Enzymatic catalysis for hydrolyzing chromogenic or fluorogenic substrates to yield a detectable signal has been used in a number of microbial diagnostic applications.
One example of this detection methodology utilizes testing for the presence of various enzymes of the targeted microorganism(s). For example, Townsend and Chen, describes a method and composition for detecting bacterial contamination in food products in U.S. application Ser. No. 08/484,593, filed Jun. 7, 1995, which is incorporated by reference herein. This growth medium detects bacterial contamination in a sample by detecting bacterial enzymes (e.g., phosphatase, βglucosidase, and L-alanine-aminopeptidase) from diverse microbial species. The liberated fluorescent moieties exhibit detectable signals with an identical emission wavelength. This procedure takes advantage of combining different bacterial enzyme activities from diverse microbes to create a broader enzyme activity spectrum; the broadened spectrum enables the detection of total bacteria in a test sample.
Koumura et. al., in U.S. Pat. No. 4,591,554 describe the use of 4-methylubelliferyl derivatives fluorogenic analysis to detect and determine the number of microorganisms based on the amount of liberated umbelliferone derivatives. According to the method, microorganisms at more than 10,000 cfu/ml can be determined by contacting a sample solution with the umbelliferone derivatives, and measuring the amount of fluorescent umbelliferone derivatives liberated. In the Koumura patent, cell lysis is required to increase the amount of liberated enzymes. In other cases, pH adjustment of the mixture and centrifugation of the mixture to remove insoluble cells are required at the end of incubation.
Edberg et. al. in U.S. Pat. No. 4,925,789 describe the use of enzyme substrates as nutrient indicators containing a nutrient moiety covalently linked to a non-metabolized detectable moiety that are specifically hydrolyzed by targeted microorganisms. In the Edberg patent, the nutrient indicators are specifically selected to be hydrolyzed by only the targeted microorganisms by virtue of their presence in the growth medium as a primary nutrient source. Non-target microorganisms are not detected in this system and are suppressed by the addition of antimicrobial agents.
While the aforementioned strategies are useful they are not particularly suited to all types of detection, especially when antimicrobial agents wish to be avoided for enhanced sensitivity. For example, most antimicrobial agents are designed to be effective against a wide spectrum of microorganisms. As a result, the use of these agents in a diagnostic test can often negatively impact the growth and detection of the target microorganism resulting in lowered sensitivity for the test. Furthermore, very few cocktails of antimicrobials are effective at suppressing all non-target growth resulting in lowered specificity for the test. In addition, while the use of a highly selective detection molecule may overcome these obstacles, such detection molecules are rare.
As it may be useful to detect any number of specific microorganisms, the prototypical example set forth for discussion purposes only is the bacterial species Campylobacter. Campylobacter species are gram-negative, oxidase-positive, curved or spiral rod-shaped bacteria. Campylobacter are a major cause of food poisoning, primarily from poultry products, and their incidence in outbreaks of gastrointestinal disease appears to be on the rise. The United States Department of Agriculture is leading the effort to reduce the levels of Campylobacter contamination on poultry products by developing methods for the detection and quantification of Campylobacter in poultry rinse water samples. In the most widely used current procedure (United States Department of Agriculture ARS, Poultry Microbiological Safety Research Unit, Method for the Enumeration of Campylobacter species from Poultry Rinses, Mar. 15, 2000; Also see, Stem et al., J. Food Prot. 55:663-666, 1992 and Stem et al., J. Food Prot. 55:514-517, 1992, incorporated by reference in their entirety)) 400 ml of buffered phosphate water (BPW) is added to a strong plastic bag containing a raw chicken carcass. The bag is twisted, and the contents shaken for 2 minutes to liberate any Campylobacter from the surface of the chicken into the BPW. After rinsing, at least 40 ml of rinse water is transferred to a sterile container and held on ice until it can be tested for the presence of Campylobacter. To quantify the Campylobacter, 0.25 ml aliquots of rinse water are spread onto four plates of agar based medium (either Campy-Line agar or Campy CEFEX agar, recipe available from United States Department of Agriculture ARS, Poultry Microbiological Safety Research Unit; also available from Hardy Diagnostics, Santa Maria, Calif.). Next, 0.1 ml aliquots of rinse water are spread onto two additional plates of medium. These six plates are then placed in a 42° C. microaerophillic incubator and incubated for 48 hours. Resulting colonies that resemble typical Campylobacter species are observed microscopically to visualize cell shape and the presence of cell motility. Typical comma-shaped cells are finally subjected to a slide agglutination test to confirm the presence of Campylobacter. 
This method, although widely accepted, has a number of disadvantages the most important of which is that the method requires a large number of complicated steps to confirm that the organisms growing on the plates are in-fact Campylobacter. As with most microorganism detection assays, these steps are very expensive to run, require the skills of a highly trained microbiologist to perform, and significantly limit the number of tests that can be performed in a normal laboratory shift. As a result, short cuts are made by the technicians performing the tests to complete their work on-time which can result in highly erroneous data, thus, affecting the quality of the food being prepared. Therefore, there is a need for improved tests for the detection of Campylobacter. If the confirmation step could be simplified, and test results obtained in a shorter more cost effective manner, it would allow manufacturers to release product with more accurate information regarding the true Campylobacter concentration. Obviously, this would represent a significant labor and cost savings for manufacturers and would also benefit the consumer by providing for safer foods.
The present invention resolves the aforementioned difficulties in the art while providing other related advantages.